Conformational stability of serine proteinase inhibitor from the sea anemone Heteractis crispa

The influence of different environmental values of the pH and temperature on the spatial organization of serine proteinase inhibitor from the sea anemone Heteractis crispa (=Radianthus macrodactylus) on the level of tertiary and secondary structure was studied by CD spectroscopy. The molecule InhVJ was shown to possess a high conformational thermo- and pH-stability. We determined the point of conformational thermotransition of polypeptide (70 degrees C) after which the molecule gets denaturational stable state with conservation of 80% proteinase inhibitory activity. The significant partial reversible changes of molecule spatial organization were established to occur at the level of tertiary structure in the process of acid-base titration in the range of pH 11.0-13.0. This can be explained by of ionization of tyrosine residues. The molecule InhVJ is conformationally stable at the low pH values (2.0). The quenching of tyrosine residues by acrylamide showed that two of these residues are accessible to the quencher in full, while the third part is available.     

Interaction of sea amemone Heteractis crispa Kunitz type polypeptides with pain vanilloid receptor TRPV1: in silico investigation

Using methods of molecular biology we defined the structures of the 31 sea anemone Heteractis crispa genes encoding polypeptides which are structurally homologous to the Kunitz proteinase inhibitor family. Identified amino acid sequences have point residue substitutions, high degree of homology with sequences of known H. crispa Kunitz family members, and represent a combinatorial library of polypeptides. We generated their three-dimensional structures by homologous modeling methods. Analysis of their molecular electrostatic potential enabled us to divide given polypeptides into three clusters. One of them includes polypeptides APHC1, APHC2 and APHC3, which were earlier shown to possess a unique property of inhibiting of the pain vanilloid receptor TRPV1 in vitro and providing the analgesic effects in vivo in addition to their trypsin inhibitory activity. Molecular docking made possible establishing the spatial structure of the complexes, the nature of the polypeptides binding with TRPV1, as well as functionally important structural elements involved in the complex formation. Structural models have enabled us to propose a hypothesis contributing to understanding the APHC1-3 impact mechanism for the pain signals transduction by TRPV1: apparently, there is an increase of the receptor relaxation time resulted in binding of its two chains with the polypeptide molecule, which disrupt the functioning of the TRPV1 and leads to partial inhibition of signal transduction in electrophysiological experiments.     

Interaction of sea anemone <Emphasis Type="Italic">Heteractis crispa</Emphasis> Kunitz type polypeptides with pain vanilloid receptor TRPV1: In silico investigation

Using methods of molecular biology we defined the structures of the 31 sea anemone Heteractis crispa genes encoding polypeptides which are structurally homologous to the Kunitz protease inhibitor family. The identified sequences have single-point amino acid substitutions, a high degree of homology with sequences of known Kunitz family members from H. crispa, and represent a combinatorial library of polypeptides. We generated their three-dimensional structures by methods of homology modeling. Analysis of their molecular electrostatic potential allowed the division of the polypeptides into three clusters. One of them includes polypeptides APHC1, APHC2, and APHC3 which have been shown to possess, in addition to their trypsin inhibitory activity, a unique property of inhibiting the pain vanilloid receptor TRPV1 in vitro and providing the analgesic effects in vivo. The spatial structure of the polypeptide complexes with TRPV1, the nature of the interactions, as well as functionally important structural elements involved in the complex formation, were established by molecular docking technique. The designed models allowed us to propose a hypothesis contributing to the understanding of how APHC1-APHC3 affect the pain signals transduction by TRPV1: apparently, relaxation time of the receptor increases due to binding of its two chains with a polypeptide molecule which disrupts functioning of TRPV1 and leads to partial inhibition of the signal transduction in electrophysiological experiments.     

瞬时受体电位香草酸亚型1(TRPV1)与炎性痛

瞬时受体电位香草酸亚型1(transient receptor potential vanilloid 1,TRPV1)是TRP超家族的成员之一,是一种非选择性的阳离子通道。TRPV1广泛分布于伤害性感受器上,并且在伤害性感受器中起重要作用。TRPV1能够感受伤害性刺激,将之转化为动作电位,传至中枢形成痛觉。炎症时释放的许多炎症介质都能够与TRPV1发生相互作用,产生疼痛或痛觉过敏,并且通过各种不同的信号通路来调制TRPV1的活性。深入研究TRPV1的作用机制,有助于理解痛觉生理和开发新型镇痛药物。     

Vertical and horizontal distribution patterns of the giant sea anemone Heteractis crispa with symbiotic anemonefish on a fringing coral reef

The distribution patterns of the leathery sea anemone, Heteractis crispa, which contains an algal endosymbiont (zooxanthellae) and anemonefish, were investigated in relation to size distribution on a shallow fringing reef (3.2 ha, 0–4 m depth) in Okinawa, Japan. Individual growth and movements were also examined. Large individuals (>1,000 cm²) inhabited reef edges up to a depth of 4 m, while small anemone (<500 cm²) inhabited shallow reefs including inner reef flats. Individuals rarely moved, and their sizes were significantly correlated with their water depths. Growth of small anemones was negatively correlated with their distance from the reef edge, suggesting that reef edges provide more prey and lower levels of physiological stress. This study suggested that deep reef edges are suitable habitats for H. crispa. Large anemones were inhabited by large Amphiprion perideraion or large Amphiprion clarkii, both of which are effective defenders against anemone predators. Anemones that settle in deep reef edges may enjoy a higher survival rate and attain a large size because of their symbiotic relationship with anemonefish. However, early settlers do not harbor anemonefish. Their mortality rate would be higher in the deep edges than in shallow edges, the complicated topography of which provides refuge.     

Intact microtubules preserve transient receptor potential vanilloid 1 (TRPV1) functionality through receptor binding

The transient receptor potential cation channel subfamily V member 1 (TRPV1) is a protein currently under scrutiny as a pharmacological target for pain management therapies. Recently, the role of TRPV1-microtubule interaction in transducing nociception stimuli to cells by cytoskeletal rearrangement was proposed. In this work, we investigate TRPV1-microtubule interaction in living cells under the resting or activated state of TRPV1, as well as in presence of structurally intact or depolymerized cytoskeletal microtubules. We combined a toolbox of high resolution/high sensitivity fluorescence imaging techniques (such as FRET, correlation spectroscopy, and fluorescence anisotropy) to monitor TRPV1 aggregation status, membrane mobility, and interaction with microtubules. We found that TRPV1 is a dimeric membrane protein characterized by two populations with different diffusion properties in basal condition. After stimulation with resiniferatoxin, TRPV1 dimers tetramerize. The tetramers and the slower population of TRPV1 dimers bind dynamically to intact microtubules but not to tubulin dimers. Upon microtubule disassembly, the interaction with TRPV1 is lost thereby inducing receptor self-aggregation with partial loss of functionality. Intact microtubules play an essential role in maintaining TRPV1 functionality toward activation stimuli. This previously undisclosed property mirrors the recently reported role of TRPV1 in modulating microtubule assembly/disassembly and suggests the participation of these two players in a feedback cycle linking nociception and cytoskeletal remodeling.     

Endogenous unsaturated C18 N-acylethanolamines are vanilloid receptor (TRPV1) agonists

The endogenous C18 N-acylethanolamines (NAEs) N-linolenoylethanolamine (18:3 NAE), N-linoleoylethanolamine (18:2 NAE), N-oleoylethanolamine (18:1 NAE), and N-stearoylethanolamine (18:0 NAE) are structurally related to the endocannabinoid anandamide (20:4 NAE), but these lipids are poor ligands at cannabinoid CB(1) receptors. Anandamide is also an activator of the transient receptor potential (TRP) vanilloid 1 (TRPV(1)) on primary sensory neurons. Here we show that C18 NAEs are present in rat sensory ganglia and vascular tissue. With the exception of 18:3 NAE in rat sensory ganglia, the levels of C18 NAEs are equal to or substantially exceed those of anandamide. At submicromolar concentrations, 18:3 NAE, 18:2 NAE, and 18:1 NAE, but not 18:0 NAE and oleic acid, activate native rTRPV(1) on perivascular sensory nerves. 18:1 NAE does not activate these nerves in TRPV(1) gene knock-out mice. Only the unsaturated C18 NAEs elicit whole cell currents and fluorometric calcium responses in HEK293 cells expressing hTRPV(1). Molecular modeling revealed a low energy cluster of U-shaped unsaturated NAE conformers, sharing several pharmacophoric elements with capsaicin. Furthermore, one of the two major low energy conformational families of anandamide also overlaps with the cannabinoid CB(1) receptor ligand HU210, which is in line with anandamide being a dual activator of TRPV(1) and the cannabinoid CB(1) receptor. This study shows that several endogenous non-cannabinoid NAEs, many of which are more abundant than anandamide in rat tissues, activate TRPV(1) and thus may play a role as endogenous TRPV(1) modulators.     

A new inhibitor of elastase from the sea anemone (Anemonia sulcata)

A proteinase inhibitor for elastases was isolated from extracts of the sea anemone Anemonia sulcata and purified to apparent homogeneity. The procedure comprises ethanolic extraction of the deep-frozen animals followed by gel filtration on Sephadex G-50 and by ion exchange chromatography on DEAE-Sephadex A-25 and SP-Sephadex C-25 and by hydroxylapatite chromatography. The slightly acidic inhibitor (isoelectric point 5.9) is a small protein consisting of 48 amino-acid residues without tryptophan and phenylalanine. The single chain molecule contains two methionines and no free sulfhydryl group but six cysteines presumably forming disulfide bonds. Reaction with cyanogen bromide abolishes the inhibitory properties. The inhibitor exhibits a rather narrow specificity for elastases. It strongly inhibits porcine pancreatic elastase in a permanent fashion with an equilibrium dissociation constant Ki of about 10(-10)M and somewhat weaker the elastase from human leucocytes with a Ki of about 10(-7)M. No obvious inhibition is observed of other serine proteinase such as bovine trypsin, bovine chymotrypsin, subtilisin from Bacillus subtilis and cathepsin G from human leucocytes when tested with synthetic substrates.     

A naturally enhanced green fluorescent protein from magnificent sea anemone (Heteractis magnifica) and its functional analysis

A novel fluorescent protein termed hmGFP homologous to the green fluorescent protein (GFP) from Aequorea victoria was cloned from the tentacles of sea anemone Heteractis magnifica by EST sequencing and analysis of cDNA library and followed by using RT-PCR. The sequence analysis suggested that the chromophore, consensus amino acids, and secondary structure of 11 beta-strands of hmGFP were similar to those of GFP from other species. The recombinant hmGFP protein with high purity was obtained by the fusion expression of pETTRX-hmGFP in Escherichia coli and subsequent purification. The pH sensitivity and fluorescence spectroscopy of recombinant hmGFP were characterized. The excitation spectrum of recombinant hmGFP has a rather wide major peak with a maximum at 490 nm and a shoulder at 420 nm, and its emission spectrum at 510 nm. The expression of hmGFP and the chimera IPL through hmGFP in CHO cells has shown that the fusion protein IPL through hmGFP has retained the normal membrane targeting of the IPL from Dasyatis akajei, as well as maintaining fluorescent properties similar to those of native hmGFP, suggesting a promising prospect of the application in biotechnology research for the new protein.     

20-Hydroxyeicosatetraenoic acid (20-HETE) is a novel activator of transient receptor potential vanilloid 1 (TRPV1) channel

TRPV1 is a member of the transient receptor potential ion channel family and is gated by capsaicin, the pungent component of chili pepper. It is expressed predominantly in small diameter peripheral nerve fibers and is activated by noxious temperatures >42 °C. 20-Hydroxyeicosatetraenoic acid (20-HETE) is a cytochrome P-450 4A/4F-derived metabolite of the membrane phospholipid arachidonic acid. It is a powerful vasoconstrictor and has structural similarities with other TRPV1 agonists, e.g. the hydroperoxyeicosatetraenoic acid 12-HPETE, and we hypothesized that it may be an endogenous ligand for TRPV1 in sensory neurons innervating the vasculature. Here, we demonstrate that 20-HETE both activates and sensitizes mouse and human TRPV1, in a kinase-dependent manner, involving the residue Ser(502) in heterologously expressed hTRPV1, at physiologically relevant concentrations.     

Synthesis and evaluation of thiazole carboxamides as vanilloid receptor 1 (TRPV1) antagonists

A thiazole derivative, 2-(2,6-dichlorobenzyl)-N-(4-isopropylphenyl) thiazole-4-carboxamide (1), was identified as a TRPV1 antagonist. We synthesized various thiazole analogs and evaluated them for their ability to block capsaicin- or acid-induced calcium influx in TRPV1-expressing CHO cells. The IC(50) values of the most potent antagonists were ca. 0.050microM in these assays.     

Trisubstituted pyrimidines as transient receptor potential vanilloid 1 (TRPV1) antagonists with improved solubility

A series of trisubstituted pyrimidines were synthesized to improve aqueous solubility of our first TRPV1 clinical candidate (1; AMG 517), while maintaining potent TRPV1 inhibitory activity. Structure-activity and structure-solubility studies led to the identification of compound 26. The aqueous solubility of 26 (>or=200microg/mL, 0.01 HCl; 6.7microg/mL, phosphate buffered saline (PBS); 150microg/mL, fasted-state simulated intestinal fluid (SIF)) was significantly improved over 1. In addition, compound 26 was found to be orally bioavailable (rat F(oral)=24%) and had potent TRPV1 antagonist activity (capsaicin IC(50)=1.5nM) comparable to that of 1.     

Embryonic and larval development of the host sea anemones Entacmaea quadricolor and Heteractis crispa

Little information is available on the sexual reproductive biology of anemones that provide essential habitat for anemonefish. Here we provide the first information on the surface ultrastructural and morphological changes during development of the embryos and planula larvae of Entacmaea quadricolor and Heteractis crispa, using light and scanning electron microscopy. Newly spawned eggs of E. quadricolor and H. crispa averaged 794 microm and 589 microm diameter, respectively, and were covered by many spires of microvilli that were evenly distributed over the egg surface, except for a single bare patch. Eggs of both species contained abundant zooxanthellae when spawned, indicating vertical transmission of symbionts. Fertilization was external, and the resulting embryos displayed superficial cleavage. As development continued, individual blastomeres became readily distinguishable and a round-to-ovoid blastula was formed, which flattened with further divisions. The edges of the blastula thickened, creating a concave-convex dish-shaped gastrula. The outer margins of the gastrula appeared to roll inward, leading to the formation of an oral pore and a ciliated planula larva. Larval motility and directional movement were first observed 36 h after spawning. E. quadricolor larval survival remained high during the first 4 d after spawning, then decreased rapidly.     

Structure-activity relationships of 1,4-dihydropyridines that act as enhancers of the vanilloid receptor 1 (TRPV1)

Vanilloid agonists such as capsaicin activate ion flux through the TRPV1 channel, a heat- and ligand-gated cation channel that transduces painful chemical or thermal stimuli applied to peripheral nerve endings in skin or deep tissues. We have probed the SAR of a variety of 1,4-dihydropyridine (DHP) derivatives as novel 'enhancers' of TRPV1 activity by examining changes in capsaicin-induced elevations in (45)Ca(2+)-uptake in either cells ectopically expressing TRPV1 or in cultured dorsal root ganglion (DRG) neurons. The enhancers increased the maximal capsaicin effect on (45)Ca(2+)-uptake by typically 2- to 3-fold without producing an action when used alone. The DHP enhancers contained 6-aryl substitution and small alkyl groups at the 1 and 4 positions, and a 3-phenylalkylthioester was tolerated. Levels of free intracellular Ca(2+), as measured by calcium imaging, were also increased in DRG neurons when exposed to the combination of capsaicin and the most efficacious enhancer 23 compared to capsaicin alone. Thus, DHPs can modulate TRPV1 channels in a positive fashion.     

New polypeptide components from the <Emphasis Type="Italic">Heteractis crispa</Emphasis> sea anemone with analgesic activity

Two new polypeptide components which exhibited an analgesic effect in experiments on mice were isolated from the Heteractis crispa sea tropical anemone by the combination of chromatographic methods. The APHC2 and APHC3 new polypeptides consisted of 56 amino acid residues and contained six cysteine residues. Their complete amino acid sequence was determined by the methods of Edman sequencing, mass spectrometry, and peptide mapping. An analysis of the primary structure of the new peptides allowed for their attribution to a large group of trypsin inhibitors of the Kunitz type. An interesting biological function of the new polypeptides was their analgesic effect on mammals, which is possibly realized via the modulation of the activity of the TRPV1 receptor and was not associated with the residual inhibiting activity towards trypsin and chymotrypsin. The analgesic activity of the APHC3 polypeptide was measured on the hot plate model of acute pain and was significantly higher than that of APHC2. Methods of preparation of the recombinant analogues were created for both polypeptides.     

4-Aminophenyl acetamides and propanamides as potent transient receptor potential vanilloid 1 (TRPV1) ligands

A series of 2-(3,5-substituted 4-aminophenyl)acetamide and propanamide derivatives were investigated as human TRPV1 antagonists. The analysis of the structure-activity relationship indicated that 2-(3,5-dihalo 4-aminophenyl)acetamide analogues displayed excellent antagonism of hTRPV1 activation by capsaicin and showed improved potency compared to the corresponding propanamides. The most potent antagonist (36) exhibited potent and selective antagonism for hTRPV1 not only to capsaicin but also to NADA and elevated temperature; however, it only displayed weak antagonism to low pH. Further studies indicated that oral administration of antagonist 36 blocked the hypothermic effect of capsaicin in vivo but demonstrated hyperthermia at that dose. A docking study of 36 was performed in our established hTRPV1 homology model to understand its binding interactions with the receptor and to compare with that of previous antagonist 1.     

Tetrahydropyridine-4-carboxamides as novel, potent transient receptor potential vanilloid 1 (TRPV1) antagonists

A series of 1,2,3,6-tetrahydropyridyl-4-carboxamides, exemplified by 6, have been synthesized and evaluated for in vitro TRPV1 antagonist activity, and in vivo analgesic activity in animal pain models. The tetrahydropyridine 6 is a novel TRPV1 receptor antagonist that potently inhibits receptor-mediated Ca2+ influx in vitro induced by several agonists, including capsaicin, N-arachidonoyldopamine (NADA), and low pH. This compound penetrates the CNS and shows potent anti-nociceptive effects in a broad range of animal pain models upon oral dosing due in part to its ability to antagonize both central and peripheral TRPV1 receptors. The SAR leading to the discovery of 6 is presented in this report.